In vitro co-culture system for investigating Armillaria root rot in Prunus spp. using a fiber-supported liquid approach

In vitro co-culture techniques that allow the growth of plants and pathogens under controlled environmental conditions are being used to re-create host plant infection. These approaches reduce infection times, promote reproducibility, and enable a rapid evaluation of plant-pathogen interactions. As a result, these systems have become essential in breeding programs aimed at developing plant resistance to diseases. In this study, we developed and validated an in vitro co-culture system to investigate the Armillaria root rot (ARR) affecting Prunus spp. This disease, caused by fungi Armillaria spp. and Desarmillaria caespitosa, poses a severe threat to the stone and nut fruit industry due to the susceptibility of most commercial rootstocks to infection and the lack of effective management options for its control. The system consists of a fiber-supported liquid approach in sterile plastic vessels that allows a fast and reproducible fungal infection under controlled environmental conditions. The floor of the vessels was covered with a polyester-fiber matte and a germination paper that served as an interface between the mycelia and the plant roots. The vessels were subjected to inoculation with Armillaria mellea and D. caespitosa, and three Prunus genotypes (‘Guardian®’, ‘MP-29’, and Prunus cerasifera ‘14–4’) were co-cultured with both fungi. Disease progression and plant and fungal biomass were monitored during co-culture. The presented in vitro co-culture approach facilitates the concurrent growth of Armillaria/Desarmillaria spp. and Prunus spp., excluding most of the limitations associated with greenhouses and field experiments. This system provides consistent and reproducible conditions for investigating a prominent plant disease affecting Prunus spp.


Materials
ethanol for 1 minute, followed by rinsing with sterile deionized water.Then, immerse the shoots in a 10% bleach solution for 10 minutes, and rinse them twice with deionized water (Figure 1).

2
Establishment of plant cultures from seeds 2.1 Clean fruit exocarps with 20% bleach for 10 minutes, followed by a 10-minute immersion in 70% ethanol.

2.2
Extract seeds within a laminar ow hood and transfer them aseptically into culture tubes containing Woody Plant Medium.Allow them to undergo strati cation for ten weeks in darkness at 4 ºC.

Establishment of Plant Cultures
Optional: When the When the presence of hyper multiplication, an occasional resting cycle with 16 μM indole-3-acetic acid (resting media) is recommended.

4
Propagate fungal cultures in Petri plates by placing two plugs (0.5 × 0.5 cm) from the youngest part of the colony (Figure 3).6

Fungi Preservation
Refresh every 14 days by transferring mycelial plugs to fresh MEA plates to ensure active fungal growth.

7
Extract three ten-millimeter-diameter plugs from the edge of two-week-old colonies 8 Remove most of the agar plug and homogenize mycelium with 5 mL of sterile water using a sterilized 15 mL tissue grinder (Figure 4).11

Inoculum preparation
Prepare the mycelium suspension for inoculum by taking a 2 × 2 cm plug from the previously prepared plate and homogenate with 7 mL of sterile water using a tissue grinder, and aliquoted in 1.5 mL Eppendorf tubes (Figure 5).12 Autoclave (121 ºC for 20 min) rectangular plastic vessels (110 × 297 mm; Southern Sun BioSystems) and after cooling down to room temperature, set the ber-supported paper on the oor of each vessel (Figure 6).
Co-culture (plant-fungi) establishment  13 Add 175 mL of plant growth regulator-free liquid 'New Prunus Medium' to each vessel.

14
Transfer fteen in vitro plants from an agar-based medium to each vessel removing the agar and sealing vessels with polyvinyl chloride lm (Figure 7).15 Place vessels on a rocker's arm with an articulated shelf that providesone swing every 15 min.

16
Use another set of rectangular Southern Sun BioSystems vessels containing 175 mL of 'New Prunus Medium', ber matte, and germination paper and add 1 mL of the mycelium suspension for inoculum.

17
Seal the vessels with PVC lm and place them on an automatic rocker arm at 5 rpm under μM/m2/s LED light 2 red 1 blue and 16 h/day photoperiod at 24 ºC.

18
After ten and seventeen days of inoculation with A. mellea and D. caespitosa, respectively, transfer the in vitro rooted plants from the liquid media to the inoculated vessels.

19
Add 60 mL of 'New Prunus Medium' without any plant growth regulator just before plant transferring.

20
Seal the vessels with PVC lm and place them on an automatic rocker arm at 5 rpm under μM/m2/s LED light 2 red 1 blue and 16 h/day photoperiod at 24 ºC (Figure 8).

21
Collect tissues when needed.

Figure 1 .
Figure 1.Cleaning process of dormant shoots

Figure 5 .
Figure 5. Preparation of the inoculum suspension for infection.

Figure 6
Figure 6.b) Southern Sun BioSystems with ber-supported paper inside.

Figure 7 .
Figure 7.In vitro plants growing in the Southern Sun BioSystems vessels.